Beta lactam antibiotic catechol derivative and preparation and application thereof

Abstract

The invention relates to the field of pharmacy, in particular to a beta lactam antibiotic catechol derivative and preparation and application thereof. The beta lactam antibiotic catechol derivative is a compound shown in the formula I, and # imgabs0. The beta lactam antibiotic catechol derivative disclosed by the invention is prepared by taking dihydroxybenzoic acid as a catechol type siderophore, and coupling the catechol type siderophore with beta lactam antibiotics; the coupled antibiotics can be transported to the acting part in a targeted manner by utilizing a unique siderophore transport system of bacteria, and the drug effect can be gradually improved aiming at the drug sensitive flora of a parent nucleus. According to the invention, the drug with drug resistance can play the drug effect again, and drug resistance caused by insufficient antibiotic transport due to factors such as bacterial surface variation is avoided.

Description

Technical Field

[0001] The invention relates to the field of pharmacy, and in particular to a beta-lactam antibiotic catechol derivative and a preparation and application thereof. Background Art

[0002] Nowadays, humans have been attacked by various infectious diseases, which can easily take the lives of millions of people. It was not until 1928 that Alexander Fleming accidentally discovered penicillin, and antibiotics, a powerful drug, officially came onto the stage of history, providing a powerful weapon against infectious diseases. However, with the abuse of antibiotics, the problem of drug resistance has continued to emerge. Most of them died from methicillin-resistant Staphylococcus aureus (MRSA), and the specific number of deaths caused by this type of resistant bacteria has exceeded 100,000. Therefore, it is necessary to find more powerful drugs to deal with the threat of multidrug-resistant bacteria.

[0003] At present, the number of multidrug-resistant bacteria (MDR), pan-drug-resistant bacteria (XDR) and even pan-drug-resistant bacteria (PDR) is increasing, which has posed a great threat to human health. Bacterial resistance is the development of a variety of unique mechanisms by bacteria to resist the killing effect of antibiotics, such as improving the efficiency of antibiotic exocytosis pumps, reducing the permeability of the cell membrane phospholipid layer, secreting antibiotic-degrading enzymes, and changing the drug target. There are certain differences in the composition of the cell wall between Gram-positive bacteria and Gram-negative bacteria. Gram-positive bacteria have a thick layer of peptidoglycan on the cytoplasmic membrane, which can protect against adverse environments, while Gram-negative bacteria lack the lipopolysaccharide (LPS) layer in the outer membrane of the cell wall. The reason why Gram-negative bacteria are more resistant to antibiotics than Gram-positive bacteria is that Gram-negative bacteria have a unique outer membrane structure. Antibiotics must be able to pass through the cell wall to reach the site of action to work. Generally, hydrophilic drugs can enter the bacteria through channel proteins, and hydrophobic drugs can enter the bacteria through free diffusion. Therefore, any changes or mutations in the outer membrane may cause antibiotics to be unable to reach the site of action and thus produce drug resistance. Take Helicobacter pylori, a major pathogen, for example. It can cause gastritis, peptic ulcers and even gastric cancer. Nowadays, the drug resistance of Helicobacter pylori is increasing. The reasons for drug resistance include the spillover of ribosomal protein L22 and the overexpression of translation initiation factor IF-2.

[0004] In order to overcome the problem of bacterial resistance caused by insufficient diffusion of drugs into bacteria, changes in bacterial surface receptors, and antibiotic efflux, the use of iron as a carrier is a way. Iron is an essential nutrient for bacterial growth. Bacteria obtain iron in the environment by secreting iron carriers. Natural iron carriers can be divided into hydroxamic acid type, catechol type, carboxylate type, and mixed type. The combined use of iron carriers and antibiotics, such as the "Trojan Horse" strategy, uses the bacterial iron absorption system to promote the endocytosis of antibiotics, thereby improving the antibacterial effect of antibiotics. The successful development of iron carrier antibiotics such as cefuroxime demonstrates the effectiveness and potential of this strategy. The catechol iron carrier has attracted much attention due to its unique properties and has great potential. First, it shows an extremely high affinity for trivalent iron ions, which enables it to effectively bind to iron and deliver it to the inside of bacteria. Secondly, this type of carrier is widely present in a variety of bacteria and strains, so it has a wide range of applicability. Finally, catechol siderophores can be chemically synthesized through a variety of pathways, which provides more possibilities for their application; however, due to factors such as bacterial surface variation, insufficient antibiotic transport leads to drug resistance, which needs to be addressed so that the drug can regain its efficacy. Summary of the invention

[0005] In view of the above problems, the purpose of the present invention is to provide a preparation and application of a β-lactam antibiotic catechol-siderophore conjugate.

[0006] To achieve the above purpose, the present invention adopts the following technology:

[0007] A β-lactam antibiotic catechol derivative, wherein the β-lactam antibiotic catechol derivative is a compound represented by formula 1,

[0008]

[0009] In the formula,

[0010] R1, R2 may be the same or different and may be selected from hydroxyl or hydrogen;

[0011] A is:

[0012]

[0013] Preferably, the β-lactam antibiotic catechol derivative is a compound represented by Formula 1, wherein R1 and R2 may be the same or different and are selected from hydroxyl or hydrogen;

[0014] A is:

[0015]

[0016] More preferably, the β-lactam antibiotic catechol derivative is:

[0017]

[0018]

[0019] A method for preparing the β-lactam antibiotic catechol derivative:

[0020] (1) Compound (I-1), compound (II) and compound (III) are reacted in N,N-dimethylformamide at a temperature from 0°C to room temperature to obtain compound (IV-1);

[0021]

[0022] (2) Compound (IV-1), antibiotics and potassium carbonate are reacted in N,N-dimethylformamide at 30°C to prepare a compound of formula 1.

[0023] In the step (1), compound (I-1), compound (II) and compound (III) are dissolved in N,N-dimethylformamide at 0°C, stirred until completely dissolved, heated to room temperature, stirred continuously for reaction at room temperature, and monitored by TLC until the reaction is complete. The compound (IV-1) is obtained after the reaction is complete, and the next step of the reaction is directly carried out; wherein the molar ratio of compound (I-1) to compound (II) is 1:1 to 1.5; the molar ratio of compound (I-1) to compound (III) is 1:1 to 1.5; the volume amount of N,N-dimethylformamide is 1.5 mL / mmol based on the molar amount of compound (I-1).

[0024] The step (2) comprises dissolving the antibiotic and potassium carbonate in N,N-dimethylformamide, rapidly adding the mixture into the reaction system of the step (1) under stirring, reacting at 30° C. under nitrogen protection, and monitoring the completion of the reaction by TLC, followed by purification to obtain the compound of formula 1; wherein the molar ratio of the antibiotic to potassium carbonate is 1:0.1 to 1; the molar ratio of the antibiotic to compound (IV-1) is 1:1 to 1.5; and the volumetric amount of N,N-dimethylformamide is 0.5-1 mL / mmol based on the molar amount of the antibiotic.

[0025] The purification is to pour the reaction solution into water, extract with ethyl acetate, collect the ethyl acetate phase, wash with saturated brine, dry with anhydrous sodium sulfate, and dry with rotary evaporation to obtain the compound shown in formula 1.

[0026] The developing solvent during TLC monitoring was a mixture of ethyl acetate, acetone, glacial acetic acid and water in a volume ratio of 5:2:2:1.

[0027] The antibiotic is amoxicillin, ampicillin, cefuroxime or ceftazidime.

[0028] The invention discloses an application of a beta-lactam antibiotic catechol derivative, and an application of the beta-lactam antibiotic catechol derivative in the preparation of an antibacterial agent for pathogenic bacteria.

[0029] The beta-lactam antibiotic catechol derivative is used in the preparation of an antibacterial agent for pathogenic bacteria of gram-negative bacteria.

[0030] The pathogenic bacteria are Escherichia coli or Helicobacter pylori.

[0031] The advantages of the present invention are:

[0032] The derivatives of the present invention have outstanding iron complexing ability and good lipophilicity in structure, and they are iron carriers coupled with antibiotics, which can expand the development path of antibiotics, so that drugs that have already developed drug resistance can regain their efficacy, and avoid drug resistance caused by insufficient antibiotic transport due to factors such as bacterial surface variation. Compared with the substances already on the market, the catechol derivatives of the present invention have good iron complexing ability. The derivatives can combine with Fe through their phenolic hydroxyl groups. 3+ It forms a stable hexadentate octahedral complex, and the stability of this structure is crucial for the absorption of iron. It has good lipophilicity and can more easily pass through the cell membrane, thereby improving the efficiency of iron uptake. It can also exert good efficacy in inhibiting Gram-negative bacteria, and is synthesized by a one-pot method, which makes the synthetic route simpler, greener and more efficient. The unique iron carrier transport system of bacteria can be used to transport catechol-derivatized antibiotics to their target sites. This method can enable drugs that have already developed resistance to regain their efficacy and can also improve the efficacy of antibiotic-sensitive bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the hydrogen spectrum of compound VI-1 provided in the examples of the present invention.

[0034] Figure 2 The iron complexing ability of compound VI-1 provided in the embodiment of the present invention; wherein 2-1 is the linear fitting of the iron ion solution, 2-2 is Fe 3+ Complexation results with derivatives.

[0035] Figure 3 The MIC value of compound VI-1 provided in an example of the present invention against Escherichia coli was determined, wherein A is AM, B is AM-34DBH, C is AM, and D is AM-23DBH.

[0036] Figure 4 This is the determination of the inhibition zone of compound VI provided in the examples of the present invention against Escherichia coli.

[0037] Figure 5 The MIC value of compound VI provided in the examples of the present invention against Helicobacter pylori was determined.

[0038] Figure 6 The MIC value of compound VI provided in the embodiment of the present invention against Escherichia coli was determined, wherein A was a bacterial solution control group, B was AM, C was AM-23DBH, D was AM-34DBH, E was AMP, F was AMP-23DBH, G was AMP-34DBH, and H was a blank control group.

[0039] Figure 7 The MIC value of compound VII provided in an embodiment of the present invention against Escherichia coli is determined; wherein, 7-1 is the MIC value determination of compounds VII-1 to VII-2 against Escherichia coli, wherein A is CZ, B is CZ-23DBH, C is CZ-34DBH, and D is a bacterial solution control group; 7-2 is the MIC value determination of compounds VII-3 to VII-4 against Escherichia coli, wherein A is a bacterial solution control group, B is CAZ, C is CAZ-23DBH, and D is CAZ-34DBH. DETAILED DESCRIPTION

[0040] The specific implementation modes of the present invention are further described below in conjunction with examples. It should be noted that the specific implementation modes described here are only for illustrating and explaining the present invention, and are not limited to the present invention.

[0041] The present invention uses dihydroxybenzoic acid as a catechol iron carrier, couples the catechol iron carrier with a β-lactam antibiotic, and obtains a β-lactam antibiotic catechol derivative, which can utilize the iron carrier transport system unique to bacteria to transport the coupled antibiotic to its site of action in a targeted manner, and can gradually improve the efficacy of bacterial flora with parent nucleus drug sensitivity. The present invention can enable drugs that have already developed drug resistance to regain their efficacy, and avoid drug resistance caused by insufficient antibiotic transport due to factors such as bacterial surface variation.

[0042] The room temperature in this aspect is 25-30°C.

[0043] Example 1: Preparation of (2S, 5R, 6R)-6-((R)-2-(3,4-dihydroxybenzamido)-2-(4-hydroxyphenyl)acetamido)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid VI-1

[0044]

[0045] (1) At 0°C, 3.08 g (20 mmol) of 3,4-dihydroxybenzoic acid, 5.75 g (30 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 3.24 g (24 mmol) of 1-hydroxybenzotriazole were added to a 100 ml round-bottom flask and dissolved in 30 mL of N,N-dimethylformamide. The mixture was stirred until completely dissolved, and the mixture was warmed to room temperature and stirred for 7 h. The reaction was monitored by TLC (developing solvent: ethyl acetate: acetone: glacial acetic acid: water = 5:2:2:1, v / v). After the reaction was completed, the intermediate 1H-benzo[d][1,2,3]triazol-1-yl 3,4-dihydroxybenzoate IV was obtained and the reaction was carried out in the next step.

[0046] (2) At 30°C, with nitrogen protection throughout the process, 8.77 g (24 mmol) of amoxicillin and 0.66 (4.8 mmol) of potassium carbonate were dissolved in 12 mL of N,N-dimethylformamide, and added to the reaction system obtained in the above step (1) with stirring. Stirring was continued for 7 h. The reaction process was monitored by TLC (developing solvent: ethyl acetate: acetone: glacial acetic acid: water = 5:2:2:1, v / v). After the reaction, the reaction solution was poured into water and extracted with ethyl acetate. The ethyl acetate phase was washed with saturated brine, dried over anhydrous sodium sulfate, and rotary evaporated to obtain 9.05 g of a light yellow solid product, namely (2S, 5R, 6R)-6-((R)-2-(3,4-dihydroxybenzamide)-2-(4-hydroxyphenyl)acetamide)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid VI, with a yield of 75.3% (see Figure 1 ).

[0047] Under the same conditions, compound (I-1) and amoxicillin (VI-1) were replaced alternately with compound (I-2) The other operations were the same as those of ampicillin to prepare compounds VII-2 to VII-4 respectively;

[0048] Under the same conditions, compound (I-1) and amoxicillin (VI-1) were replaced alternately with compound (I-2) Ceftizoxime and ceftazidime were used in the same manner to prepare compounds VIII-2 to VIII-4, respectively.

[0049] Depend on Figure 1 It can be seen that the structure of the compound is consistent with the expected one.

[0050] Example 2: Study on the iron ion complexing ability of compound VI-1

[0051] (1) Preparation of ferric chloride stock solution: Accurately weigh ferric chloride and dissolve it in 9.75 mL of purified water and 0.25 mL of 12 M hydrochloric acid to prepare a 1 mmol / mL ferric chloride stock solution.

[0052] (2) The ferric chloride stock solution was diluted with purified water to concentrations of 1.00, 0.75, 0.50, 0.25, and 0.13 mmol / mL, respectively, and the ultraviolet absorption wavelength was measured at 516 nm.

[0053] (3) Accurately weigh 1 mg of compound (VI-1), 1 mg of compound (V) and 1 mg of compound (I-1), dilute each with 1 mL of purified water, add 50 μL of compound (VI-1) solution and 150 μL of 1.00 mmol / mL ferric chloride stock solution to a 96-well plate. Under the same conditions, replace compound (VI-1) with amoxicillin (compound V) and compound (I-1) to conduct a compound VI-1 iron complexation experiment. Under the same conditions, compound (VI-1) was used to replace compound VI-2 to VI-4, amoxicillin (compound V) was replaced by ampicillin, and compound (I-1) was replaced by compound (I-2) to conduct iron complexation experiments on compound VI-2 to VI-4; under the same conditions, compound (VI-1) was used to replace compound VII-1 to VII-4, amoxicillin (compound V) was replaced by ceftazidime and ceftazidime, and compound (I-1) was replaced by compound (I-2) to conduct iron complexation experiments on compound VII-1 to VII-4.

[0054] Observe whether there is any color change in the system. If there is, the sample has the same color as Fe 3+ Under the same conditions, Fe 3+ The solution was the control group.

[0055] (4) Table 1 Compounds VI-1, V, I-1 and Fe 3+ Absorbance after complexation

[0056]

[0057] (5) Experimental conclusion:

[0058] Compared with the control group Fe 3+ In contrast, the absorbance and color of compound (VI-1) changed significantly, as shown in Table 1. Figure 2-1 and Figure 2-2 (The first row from left to right is VI-1, V, I-1; the second row is a mixture of the first and third rows, and the third row is blank Fe 3+ solution), proving that compound (V) has complex Fe after coupling with compound (I-1). 3+ ability.

[0059] Experimental Example 3: Determination of MIC value of compound VI-1 against Escherichia coli

[0060] (1) Cleaning of equipment:

[0061] Wash and wrap test tubes, conical flasks, pipette tips and other experimental equipment, sterilize them under high pressure at 121℃ for 20min, and place them on a clean bench for later use.

[0062] (2) Preparation of ID-CAMHB liquid culture medium:

[0063] Weigh appropriate amounts of the corresponding substances and dissolve them in deionized water to prepare 10 mmol / mL Zn 2+ Mg 2+ , Ca 2+ ion stock solution. According to the instructions for use of CAMHB, weigh and dilute with water to prepare 500mL of liquid culture medium. Add 50g of cationic resin to 500mL of autoclaved CAMHB to remove cations in the solution, stir at room temperature for 2h, and filter using a 0.22μm microporous filter membrane. Add an appropriate amount of ion stock solution to make Ca 2+ Concentration up to 25mg / L, Mg 2+ The concentration reaches 12.5mg / L, Zn 2+ The concentration reaches 1.0 mg / L. The pH of the culture medium is ensured to be in the range of 7.3±0.1, thereby obtaining ID-CAMHB liquid culture medium.

[0064] (3) MIC determination of samples against Escherichia coli:

[0065] Compound (VI-1) and amoxicillin (Compound V) were dissolved in DMSO and diluted to 64 μg / mL as drug solution. The MIC was determined by turbidimetry. The specific operation method was to use a pipette to take the E. coli ID-CAMHB liquid culture medium and add it to the blank ID-CAMHB liquid culture medium (add the ID-CAMHB medium with E. coli to the medium without E. coli), and dilute it to the same concentration as the turbidity of the 0.5 McFarland turbidimeter tube. At this time, the bacterial concentration in the liquid culture medium is close to 1.5×10 8 Bacteria / mL. Add 100μL of drug solution and 100μL of bacterial solution to a 96-well plate, and then dilute it step by step from left to right. Observe whether the solution grows bacteria after 24 hours. Turbidity indicates bacteria growth, and clarity indicates antibacterial activity. Use blank liquid culture medium as the blank control group and amoxicillin (Compound V) as the positive control group. Make 2 parallel groups for each sample.

[0066] (4) Experimental conclusion:

[0067] Compared with the positive control group, compound (VI-1) had significantly enhanced antibacterial ability against Escherichia coli. Figure 3The MIC value of compound (VI-1) was reduced to 1 / 10 to 1 / 20 of the MIC value of amoxicillin (compound V). This proves that compound (VI-1) significantly improves its efficacy against bacteria by utilizing the iron transfer pathway under iron deficiency conditions.

[0068] Experimental Example 4: Determination of the inhibition zone of compound VI-1 against Escherichia coli

[0069] (1) Preparation of ID-CAMHB solid culture medium:

[0070] This example uses the ID-CAMHB liquid culture medium prepared in Example 3. 200 mL of the liquid culture medium is taken, 4 g of agar is added, and the liquid culture medium is dissolved in a 70° C. water bath, and then sterilized at 121° C. for 20 min.

[0071] (2) Determination of the inhibition zone of samples against Escherichia coli:

[0072] The pre-added bacteria pour plate method is adopted. The specific operation method is to pour the Escherichia coli suspension prepared to the Max Planck concentration into the solid culture medium that has been cooled to room temperature in advance, and evenly spread it on the culture surface to obtain the Escherichia coli culture dish for use.

[0073] The antibacterial zone was determined by the drug sensitivity test paper method. The specific operation method was to place the drug sensitivity test paper with tweezers into the drug solution to be tested, and soak it in the solution with a concentration of 64μg / mL for 1h, take it out and dry it, and then place it on the solid culture medium with bacteria. Observation results: if the culture medium can be clearly seen, it is antibacterial, and if there is colony growth, it is not antibacterial (see Table 2); at the same time, compound (V) was used as the positive control group.

[0074] Table 2 Antibacterial effect of compound VI

[0075]

[0076] (3) Experimental conclusion:

[0077] Compared with the inhibition zones of amoxicillin (Compound V) and compound (VI-1) against Escherichia coli, the antibacterial ability of compound (VI-1) against Escherichia coli was significantly enhanced. Figure 4 (AM-34DBH is compound VI-1, AM is antibiotic, 3,4-DBH is Ⅰ-1) and Table 2 prove that the efficacy of compound (VI-1) is better than that of amoxicillin (compound V).

[0078] Experimental Example 5: Determination of MIC value of compound VI-1 against Helicobacter pylori

[0079] (1) Cleaning of equipment:

[0080] Wash and wrap test tubes, conical flasks, pipette tips and other experimental equipment, sterilize them under high pressure at 121℃ for 20min, and place them on a clean bench for later use.

[0081] (2) Preparation of bovine heart and brain powder liquid culture medium:

[0082] Take 7.7g of bovine heart and brain infusion medium powder and dissolve it in 200mL of water. After autoclaving, add 10% calf serum. Add selective antibiotics to a concentration of 6mg / L vancomycin, 2mg / L amphotericin B, and 5mg / L trimethoprim.

[0083] (3) MIC determination of samples against Helicobacter pylori:

[0084] Compound (VI-1) and amoxicillin (Compound V) were dissolved in DMSO and diluted to 64 μg / mL. The MIC was determined by turbidimetry. The specific operation method was to use a pipette to take a small amount of Helicobacter pylori bovine heart and brain extract powder liquid culture medium and add it to the blank bovine heart and brain extract powder liquid culture medium, and dilute it to the same concentration as the turbidity of 0.5 McFarland turbidimetric tube. At this time, the bacterial concentration in the liquid culture medium was close to 1.5×10 8 Bacteria / mL. Add 100μL of drug solution and 100μL of bacterial solution to a 96-well plate, and then dilute it step by step from left to right. Observe whether the solution grows bacteria after 24 hours. Turbidity indicates bacteria growth, and clarity indicates antibacterial activity. Use blank liquid culture medium as the blank control group and amoxicillin (Compound V) as the positive control group. Make 2 parallel groups for each sample.

[0085] (4) Experimental conclusion:

[0086] Compared with amoxicillin (Compound V), compound (VI-1) has enhanced antibacterial ability against Helicobacter pylori, and the MIC molar ratio is reduced by 30%. Figure 5 , 24-72h, the MIC of compound (VI-1) increased to 8nmol / mL, and the MIC of amoxicillin (compound V) was 21.9nmol / mL, and the efficacy was increased by 3 times, proving that compound (VI-1) has a long-term inhibitory effect on Helicobacter pylori.

[0087] Experimental Example 6: Determination of MIC value of compound VI against Escherichia coli

[0088] (1) Cleaning of equipment:

[0089] Wash and wrap test tubes, conical flasks, pipette tips and other experimental equipment, sterilize them under high pressure at 121℃ for 20min, and place them on a clean bench for later use.

[0090] (2) Preparation of ID-CAMHB liquid culture medium:

[0091] Weigh appropriate amounts of the corresponding substances and dissolve them in deionized water to prepare 10 mmol / mL Zn 2+ Mg 2+ , Ca 2+ ion stock solution. Weigh and dilute to prepare 500mL of liquid culture medium according to the instructions for use of CAMHB. Add 50g of chelating resin to 500mL of autoclaved CAMHB to remove cations in the solution, stir at room temperature for 2h, and filter using a 0.22μm microporous filter membrane. Add an appropriate amount of ion stock solution to make Ca 2+ Concentration up to 25mg / L, Mg 2+ The concentration reaches 12.5mg / L, Zn 2+ The concentration reached 1.0 mg / L. The pH of the culture medium was maintained within the range of 7.3 ± 0.1.

[0092] (3) MIC determination of samples against Escherichia coli:

[0093] Compound (VI) and amoxicillin (Compound V) were dissolved in DMSO and diluted to 64 μg / mL. The MIC was determined by turbidimetry. The specific operation method was to use a pipette to take a small amount of E. coli ID-CAMHB liquid culture medium and add it to the blank ID-CAMHB liquid culture medium, and dilute it to the same concentration as the turbidity of the 0.5 McFarland turbidimeter tube. At this time, the bacterial concentration in the liquid culture medium was close to 1.5×10 8 Bacteria / mL. Add 100μL of drug solution and 100μL of bacterial solution to a 96-well plate, and then dilute it step by step from left to right. Observe whether the solution grows bacteria after 24 hours. Turbidity indicates bacteria growth, and clarity indicates antibacterial activity. Use blank liquid culture medium as the blank control group and amoxicillin (Compound V) as the positive control group. Make 2 parallel groups for each sample.

[0094] Under the same conditions, amoxicillin (Compound V) was replaced with ampicillin to perform MIC determination of Escherichia coli.

[0095] Compound (IV) includes compounds IV-1 to IV-4. Compounds IV-1 to IV-2 were compared with amoxicillin; compounds IV-3 to IV-4 were compared with ampicillin.

[0096] (4) Experimental conclusion:

[0097] Compared with the positive control group, compound (VI) had significantly enhanced antibacterial ability against Escherichia coli. Figure 6The MIC value of compound (VI-1) was reduced to 1 / 16 of that of amoxicillin; the MIC value of compound (VI-2) was reduced to 1 / 8 of that of amoxicillin; the MIC values ​​of compounds VI-3 to VI-4 were all reduced to 1 / 8 of that of ampicillin. This proves that compound (VI) significantly improves its efficacy against bacteria by utilizing the iron transfer pathway under iron deficiency conditions.

[0098] Experimental Example 7: Determination of MIC value of compound VII against Escherichia coli

[0099] (1) Cleaning of equipment:

[0100] Wash and wrap test tubes, conical flasks, pipette tips and other experimental equipment, sterilize them under high pressure at 121℃ for 20min, and place them on a clean bench for later use.

[0101] (2) Preparation of ID-CAMHB liquid culture medium:

[0102] Weigh appropriate amounts of the corresponding substances and dissolve them in deionized water to prepare 10 mmol / mL Zn 2+ Mg 2+ , Ca 2+ ion stock solution. Weigh and dilute to prepare 500mL of liquid culture medium according to the instructions for use of CAMHB. Add 50g of chelating resin to 500mL of autoclaved CAMHB to remove cations in the solution, stir at room temperature for 2h, and filter using a 0.22μm microporous filter membrane. Add an appropriate amount of ion stock solution to make Ca 2+ Concentration up to 25mg / L, Mg 2+ The concentration reaches 12.5mg / L, Zn 2+ The concentration reached 1.0 mg / L. The pH of the culture medium was maintained within the range of 7.3 ± 0.1.

[0103] (3) MIC determination of samples against Escherichia coli:

[0104] Compound (VII) and ceftazidime or ceftazidime were dissolved in DMSO and diluted to 64 μg / mL. The MIC was determined by turbidimetry. The specific operation method was to use a pipette to take a small amount of Escherichia coli ID-CAMHB liquid culture medium and add it to the blank ID-CAMHB liquid culture medium, and dilute it to the same concentration as the turbidity of the 0.5 McFarland turbidimeter tube. At this time, the bacterial concentration in the liquid culture medium was close to 1.5×10 8 Bacteria / mL. Add 100μL of drug solution and 100μL of bacterial solution to a 96-well plate, and then dilute it step by step from left to right. Observe whether the solution grows bacteria after 24 hours. Turbidity indicates growth of bacteria, and clarity indicates inhibition of bacteria. Use blank liquid culture medium as blank control group, ceftazidime or ceftazidime as positive control group, and make 2 parallel groups for each sample.

[0105] (4) Experimental conclusion:

[0106] Compared with the positive control group, compound (VII) showed significantly enhanced antibacterial ability against Escherichia coli. Figure 7 The MIC value of compound VII-1 was 2 times higher than that of ceftazidime, and the MIC value of compound VII-2 was not much different from that of ceftazidime; the MIC values ​​of compounds VII-3 to VII-4 were all reduced to 1 / 4 of that of ceftazidime. This proves that compound (VII) significantly improves its efficacy against bacteria by utilizing the iron transfer pathway under iron deficiency conditions.

[0107] In summary, the compounds of the present invention have novel chemical structures, especially the amoxicillin derivatives obtained by coupling amoxicillin with 3,4-dihydroxybenzoic acid-type siderophore, which have significantly better in vitro anti-Gram-negative bacterial activity than the drug amoxicillin. Other β-lactam antibiotic derivatives also have better in vitro anti-Gram-negative bacterial activity than their parent core drugs. Therefore, the compounds of the present invention can be used to treat Gram-negative bacterial infectious diseases.

Claims

1. A β-lactam antibiotic catechol derivative, characterized in that: The β-lactam antibiotic catechol derivative is a compound shown in formula 1, In the formula, R1, R2 may be the same or different and may be selected from hydroxyl or hydrogen; A is:

2. The β-lactam antibiotic catechol derivative according to claim 1, characterized in that: The β-lactam antibiotic catechol derivative is a compound shown in Formula 1, In the formula, R1 and R2 may be the same or different and are selected from hydroxyl or hydrogen; A is:

3. The β-lactam antibiotic catechol derivative according to claim 2, characterized in that: The β-lactam antibiotic catechol derivative is:

4. A method for preparing the β-lactam antibiotic catechol derivative according to claim 1, characterized in that: (1) Compound (I), compound (II) and compound (III) are reacted in N,N-dimethylformamide at a temperature from 0°C to room temperature to prepare compound (IV); (2) Compound (IV), antibiotics and potassium carbonate are reacted in N,N-dimethylformamide at 30°C to prepare a compound of formula 1.

5. The method for preparing the β-lactam antibiotic catechol derivative according to claim 4, characterized in that: In the step (1), compound (I), compound (II) and compound (III) are dissolved in N,N-dimethylformamide at 0°C, stirred until completely dissolved, heated to room temperature, stirred continuously for reaction at room temperature, and the completion of the reaction is monitored by TLC. When the reaction is completed, compound (IV) is obtained, and the next step of reaction is directly carried out; wherein the molar ratio of compound (I) to compound (II) is 1:1-1.5; the molar ratio of compound (I) to compound (III) is 1:1-1.5; the volume amount of N,N-dimethylformamide is 1.5 mL / mmol based on the molar amount of compound (I).

6. The method for preparing the β-lactam antibiotic catechol derivative according to claim 4, characterized in that: The step (2) comprises dissolving the antibiotic and potassium carbonate in N,N-dimethylformamide, rapidly adding the mixture into the reaction system of the step (1) under stirring, reacting at 30° C. under nitrogen protection, and monitoring the completion of the reaction by TLC, followed by purification to obtain the compound of formula 1; wherein the molar ratio of the antibiotic to potassium carbonate is 1:0.1-1; the molar ratio of the antibiotic to compound (IV) is 1:1-1.5; and the volumetric amount of N,N-dimethylformamide is 0.5-1 mL / mmol based on the molar amount of the antibiotic.

7. The method for preparing the β-lactam antibiotic catechol derivative according to claim 6, characterized in that: The purification is to pour the reaction solution into water, extract with ethyl acetate, collect the ethyl acetate phase, wash with saturated brine, dry with anhydrous sodium sulfate, and dry with rotary evaporation to obtain the compound shown in formula 1.

8. The method for preparing the β-lactam antibiotic catechol derivative according to claim 5 or 6, characterized in that: The developing solvent during TLC monitoring was a mixture of ethyl acetate, acetone, glacial acetic acid and water in a volume ratio of 5:2:2:

1.

9. An application of a β-lactam antibiotic catechol derivative, characterized in that: The application of the beta-lactam antibiotic catechol derivative in the preparation of antibacterial agents for pathogenic bacteria.

10. The β-lactam antibiotic catechol derivative according to claim, characterized in that: The beta-lactam antibiotic catechol derivative is used in the preparation of an antibacterial agent for pathogenic bacteria of gram-negative bacteria.

Images